title: Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia.
abstract: BACKGROUND
  A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.
  
  
  METHODS
  Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.
  
  
  RESULTS
  The second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.
  
  
  CONCLUSIONS
  Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.
authors.affiliation: Regenerative Medicine Institute and Department of Medicine, National University of Ireland, Galway and Galway University Hospital, University Road, Galway, Ireland.
authors.forename: Lisa
authors.initials: L
authors.lastname: McGinley
authors.forename: Jill
authors.initials: J
authors.lastname: McMahon
authors.forename: Padraig
authors.initials: P
authors.lastname: Strappe
authors.forename: Frank
authors.initials: F
authors.lastname: Barry
authors.forename: Mary
authors.initials: M
authors.lastname: Murphy
authors.forename: Daniel
authors.initials: D
authors.lastname: O'Toole
authors.forename: Timothy
authors.initials: T
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D003847:Deoxyglucose / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D006019:Glycolysis / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007511:Ischemia / Q000378:metabolism / Q000473:pathology; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008954:Models, Biological; D051381:Rats; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes / Q000502:physiology
chemical_list: D018840:HSP70 Heat-Shock Proteins; D049452:Green Fluorescent Proteins; D003847:Deoxyglucose